Molecular Connections between DNA Replication and Cell Death in ?-Amyloid-Treated Neurons

Background: Ectopic cell cycle reactivation in neurons is associated with neuronal death in Alzheimer's disease. In cultured rodent neurons, synthetic ?-amyloid (A?) reproduces the neuronal cell cycle re-entry observed in the Alzheimer's brain, and blockade of the cycle prevents A?-induced neurodegeneration. DNA polymerase-?, whose expression is induced by A?, is responsible for the DNA replication process that ultimately leads to neuronal death, but the molecular mechanism(s) linking DNA replication to neuronal apoptosis are presently unknown. Aim: To explore the role of a conserved checkpoint pathway started by DNA replication stress, namely the ATM-ATR/Claspin/Chk-1 pathway, in switching the neuronal response from DNA replication to apoptosis. Methods: Experiments were carried out in cultured rat cortical neurons challenged with toxic oligomers of A? protein. Results: Small inhibitory molecules of ATM/ATR kinase or Chk-1 amplified A?-induced neuronal DNA replication and apoptosis, as they were permissive to the DNA polymerase-? activity triggered by A? oligomers. Claspin, i.e., the adaptor protein between ATM/ATR kinase and the downstream Chk-1, was present on DNA replication forks of neurons early after A? challenge, and decreased at times coinciding with neuronal apoptosis. The caspase-3/7 inhibitor I maintained overtime the amount of Claspin loaded on DNA replication forks and, concomitantly, reduced neuronal apoptosis by holding neurons in the S phase. Moreover, a short phosphopeptide mimicking the Chk-1-binding motif of Claspin was able to prevent A?-challenged neurons from entering apoptosis. Conclusion: We speculate that, in the Alzheimer's brain, Claspin degradation by intervening factors may precipitate the death of neurons engaged into DNA replication.